Despite the longstanding recognition that a variety of malignancies are associated with activation of coagulation, relatively little is known about the role of specific coagulation proteases in cancer metastasis. Recently, it has become clear that factors VIIa, Xa, and thrombin may have potent and distinct effects on tumor cell growth, signalling, and metastasis, implying important non-coagulant functions for these related serine proteases. We have isolated, cloned, and expressed a specific low MW inhibitor of coagulation factor Xa, called Ancylostoma caninum Anticoagulant Peptide (AcAP), from extracts of hookworms, aggressive bloodfeeding intestinal nematodes. Using a SCID mouse model, a single subcutaneous injection of recombinant AcAP (rAcAP) was completely protective against pulmonary metastases following tail vein injection of 10- LOX human melanoma tumor cells. This dramatic in vivo effect suggests that factor Xa may play an important and specific role in melanoma metastasis. Moreover, as an inhibitor of this coagulation protease, rAcAP represents a potentially valuable therapeutic agent for inhibiting tumor metastasis in vivo. Work is planned to evaluate the mechanism of rAcAP's anti-metastatic activity, as well as the role of factor Xa in tumor cell growth, signalling, and metastasis. Using both in vitro and in vivo assays, Phase I experiments will be aimed at preclinical development of rAcAP, focusing on characterizing the spectrum of its anti-tumor activity. The methods developed in Phase I will allow for subsequent screening of a variety of malignant cell lines for factor Xa binding, in order to identify potential rAcAP-sensitive tumors. PROPOSED COMMERCIAL APPLICATION: The goal of this research is to characterize the anti-metastatic properties of a novel coagulation protease inhibitor. We hope to develop AcAP as a therapeutic agent for the treatment of a variety of cancers that are associated with activation of coagulation.